The present invention relates to polyspecific binding molecules, as well as methods of making and using such molecules. In one aspect, the invention features single-chain polyspecific binding molecules that can damage or destroy target cells. The invention is useful for a variety of applications including use in associating cells that express a T-cell receptor or an antibody binding domain.
There has been recognition that immune system cells and particularly cytotoxic T lymphocytes (CTLs) can be used to detect tumor associated antigens (TAAs). For example, CTLs derived from melanomas have been used to identify a variety of melanoma-specific antigens. See e.g., Bruggen et al., Science, (1991), 254:1643; Bakker et al., J. Exp. Med., (1994), 179: 1005; and Yanuck et al., Cancer Research, (1993), 53, 3257.
Several anti-tumor therapies have attempted to use CTLs to treat diseases such as cancer. In one approach, anti-tumor CTLs are taken from a patient, expanded in vitro, and then given back to the patient to treat the cancer. However, this approach suffers from significant drawbacks. For example, it is not always straightforward to isolate sufficient quantities of the CTLs from the patient. In addition, at least some of the CTLs may have specificities that have survived self-tolerance that could lead to additional complications. See, e.g., Browning et al., Curr. Opin. Immunol., (1992) 4, 613; Mizoguchi et al., Science, (1992), 258:1795, and George et al., J. Immunol., (1994), 152, 1802.
There have been attempts to mitigate these and other shortcomings by making and using recombinant immune molecules such as those resembling antibodies. An antibody has a recognized structure that includes an immunoglobulin heavy and light chain. The heavy and light chains include an N-terminal variable region (V) and a C-terminal constant region (C). The heavy chain variable region is often referred to as xe2x80x9cVHxe2x80x9d and the light chain variable region is referred to as xe2x80x9cVLxe2x80x9d. The VH and VL chains form a binding pocket that has been referred to as F(v). See generally Davis Ann. Rev. of Immunology (1985), 3: 537; and Fundamental Immunology 3rd Ed., W. Paul Ed. Raven Press LTD. New York (1993).
Recombinant antibody molecules have been disclosed. For example, several recombinant bispecific antibody (bsFv) molecules have been reported. Most of the bsFv molecules include a F(v) formatted as a single-chain (sc-Fv). More particular sc-Fv molecules include a VH linked to a VL through a peptide linker sequence. See e.g., Huston et al. PNAS (USA), (1988), 85:5879; Bird et al., Science, (1988), 242: 423; WO 94/29350; and U.S. Pat. No. 5,455,030.
Additional bsFv molecules have been disclosed. For example, some bsFv molecules have been reported to bind a T-cell protein termed xe2x80x9cCD3xe2x80x9d and a TAA. There is recognition that binding of the bsFv may facilitate an immune system response. See e.g., Jost, C. R. (1996) Mol. Immunol. 33: 211; Lindhofer, H. et al. (1996) Blood, 88: 465 1; Chapoval, A. I. et al. (1995) J. of Hematotherapy, 4: 571.
There have been attempts to develop straightforward methods of making bispecific antibody molecules. However, many of these attempts have been associated with problems. For example, many of the molecules are reported to be insoluble especially in bacterial expression systems. See e.g., Wels et al., (1992), Biotechnology, 10:1128.
Attempts to make other recombinant immune molecules have been reported. For example, there have been specific attempts to manipulate T-cell receptors (TCRs). The TCR is a membrane bound heterodimer consisting of an xcex1 and xcex2 chain that resembles an immunoglobulin variable (V) and constant (C) region. The TCR xcex1 chain includes a covalently linked V-xcex1 and C-xcex1chain. The TCR xcex2 chain includes a Vxcex2 chain covalently linked to a C-xcex2chain. See generally Davis, supra.
There have been specific efforts to manipulate the TCR by recombinant DNA techniques. For example, in one approach, the TCR has been formatted as a single-chain fusion protein comprising the TCR V regions (sc-TCR). The sc-TCR molecule has been reported to have several important uses. See e.g., Soo Hoo, W. F. et al. PNAS (USA) 89, 4759 (1992); Wxc3xclfing, C. and Plxc3xcckthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); PCT WO 96/13593; PCT WO 96/18105; and Schlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996).
The prior recombinant immune molecules are believed to be associated with significant shortcomings.
For example, there has been recognition that many tumor antigens are xe2x80x9cshedxe2x80x9d from cells, thereby providing sites for non-specific immune molecule binding. In particular, it has been proposed that many bsFv molecules inadvertently interact with the shed antigens, thereby reducing tumor cell killing efficiency.
The prior immune molecules suffer from additional drawbacks. For example, there has been recognition that many bsFv molecules cannot bind potential target antigens such as certain peptides on the surface of tumor cells. As an illustration, the tumor related protein p53 is usually not expressed on tumor cells as an intact protein. Instead, p53 has been reported to be processed and presented as a peptide in the context of a cell surface class I or class II molecule. Thus, in settings in which binding to specific cell surface peptides is needed, it has been difficult or impossible for bsFv molecules.
Further, it has been difficult to isolate some bsFv molecules without significant isolation and/or re-folding steps. See e.g., Jost, C. R. et al. supra and references cited therein.
Preparation and use of many sc-TCRs has also been associated with problems. For example, several prior methods for making the sc-TCRs have yielded insoluble and improperly folded molecules. Several strategies have been developed in an attempt to improve sc-TCR yields. However, the sc-TCRs produced by these methods often require time-consuming manipulations to obtain even modest amounts of protein. See e.g., Ward, E. S. et al. supra; Schlueter, C. J. supra; and published PCT applications WO 96/18105 and WO 96/13593.
There is a need therefore for recombinant immune molecules and particularly single-chain polyspecific binding molecules that can damage or eliminate (kill) target cells in vitro and in vivo. It would be desirable to have methods for making the polyspecific binding molecules with a minimum of difficult preparative steps.
The present invention relates to novel immune molecules and particularly to single-chain polyspecific binding proteins that damage or eliminate (kill) desired target cells. The single-chain polyspecific binding proteins include at least one receptor domain capable of specifically binding a peptide bound (loaded) to a major histocompatibility complex (MHC) or a human-leukocyte-associated antigen (HLA); and at least one antibody domain capable of binding an antigen. The present single-chain polyspecific binding molecules are fully soluble and can be isolated in significant quantities with a minimum of difficult preparative steps. Also provided are methods and compositions for screening the single-chain polyspecific binding proteins for capacity to bind desired cells.
We have made novel polyspecific binding molecules that feature a wide variety of useful activities. For example, the single-chain polyspecific binding proteins can associate cells expressing the peptide bound (loaded) MHC (HLA) to cells expressing the antigen. In most instances, the MHC (HLA) and the antigen will be on separate cells. Association of the cells in accord with the invention preferably facilitates an immune response that can damage or kill the cells expressing the peptide bound (loaded) MHC (HLA) complexes. The present invention has a wide spectrum of useful applications including use in the treatment of certain cancers and viral infections.
More particularly, the present invention features single-chain polyspecific binding proteins that include at least one single-chain T-cell receptor (sc-TCR) or functional fragment thereof sufficient to bind a particular peptide bound (loaded) to the MHC (HLA). A cell expressing the peptide bound (loaded) MHC or HLA will often be referred to herein as a xe2x80x9ctarget cellxe2x80x9d or similar term. The polyspecific binding proteins further include at least one antibody binding domain and particularly a single-chain antibody or functional fragment thereof, which antibody binding domain is sufficient to bind the antigen. In most embodiments, the antigen bound by the antibody binding domain will be expressed on a cell surface, usually on the surface of an immune cell. In particular embodiments, the antigen will be selective for the immune cells. More preferred single-chain polyspecific binding molecules of this invention are capable of forming a specific binding complex (xe2x80x9cbridgexe2x80x9d) between the peptide bound (loaded) MHC or HLA on the target cell and the antigen on the immune cell. Without wishing to be bound to theory, it is believed that formation of the bridge in accord with the invention facilitates an immune response that can damage or kill the target cells.
Preferred polyspecific binding molecules of this invention specifically bind MHC or HLA complexes. Unless otherwise specified, the term MHC and HLA as used herein means a complex to which a particular peptide is bound (loaded). In some instances, the MHC (HLA) complexes will be referenced as xe2x80x9cpMHCxe2x80x9d, xe2x80x9cpHLAxe2x80x9d or like term to denote the peptide binding (loading). The polyspecific binding molecules are thus useful for binding the MHC and HLA complexes and for bridging those complexes to an immune cell expressing a desired antigen. In some instances, the immune cell antigen bound by a particular polyspecific binding molecule will be referred to as an xe2x80x9cactivationxe2x80x9d molecule or marker to denote preferred activation of the immune cell following binding by the polyspecific molecule.
Accordingly, in one aspect, the present invention features single-chain polyspecific binding proteins that include at least one sc-TCR covalently linked (i.e. fused) to at least one single-chain antibody (sc-Ab). In embodiments in which the single-chain polyspecific molecule include one sc-TCR and one sc-Ab, the sc-TCR and the sc-Ab molecules may be directly fused together although it is generally preferred to separate the sc-TCR and sc-Ab from each other through a suitable (first) peptide linker sequence. Alternatively, functional fragments of the sc-TCR and/or sc-Ab molecules may be employed in the proteins. In preferred embodiments, the polyspecific binding proteins will include the sc-TCR linked to the sc-Ab through the first peptide linker sequence.
More particularly, the sc-TCR is preferably a single-chain V chain. The V chain will typically include a Vxcex1,xcex2 sequence in which a V-xcex1 chain is fused to a V-xcex2 chain. In a specific embodiment, the fusion is achieved by covalently linking the molecules through a (second) peptide linker sequence. The fusion product may be further covalently linked through the V-xcex1 or V-xcex2 chain to an immunoglobulin constant chain (Ig-CL) or fragment thereof if desired.
In a more specific embodiment, the C-terminus of the sc-TCR V-xcex1 chain is covalently linked by the second peptide linker sequence to the N-terminus of V-xcex2 chain. Alternatively, the C-terminus of the sc-TCR V-xcex2 chain can be covalently linked by the second peptide linker sequence to the N-terminus of the V-xcex1 chain.
In another embodiment, a TCR C-xcex2 chain or fragment thereof is covalently linked between the C-terminus of the sc-TCR V-xcex2 chain and the N-terminus of the first peptide linker sequence. Alternatively, the TCR C-xcex2 chain or the fragment may be covalently linked between the C-terminus of the sc-TCR V-xcex1 chain and the N-terminus of the first peptide linker sequence.
In another embodiment, a TCR C-xcex1 chain or fragment thereof is covalently linked between the C-terminus of the sc-TCR V-xcex1 chain and the N-terminus of the second peptide linker sequence fused to the V-xcex2 sequence. Alternatively, the C-xcex1 chain or fragment can be covalently linked between the C-terminus of the V-xcex2 chain and the N-terminus of the second peptide linker sequence fused to the V-xcex1 sequence.
In a particular embodiment, the sc-TCR includes the TCR C-xcex1 chain or fragment covalently linked between the C-terminus of the sc-TCR V-xcex1 chain and the N-terminus of the second peptide linker sequence fused to the sc-TCR V-xcex2 sequence. Further, the TCR C-xcex2 chain or fragment is covalently linked between the C-terminus of the V-xcex2 chain and the N-terminus of the first peptide linker sequence.
As discussed, the polyspecific binding molecules of this invention include at least one sc-Ab. In a particular embodiment the antibody binding domain includes at least one sc-Fv. More preferred single-chain polyspecific binding proteins include one sc-Fv or a functional fragment thereof. An illustrative sc-Fv includes at least two immunoglobulin chains and especially two immunoglobulin variable chains, e.g., a light chain (VL) fused to a heavy chain (VH). In this embodiment, the VL and VH chains may be fused together although it is generally preferred to covalently link the chains through a (third) peptide linker sequence.
In a particular embodiment, the C-terminus of the VL chain is covalently linked by the third peptide linker sequence to the N-terminus of VH chain. In another embodiment, the C-terminus of the VH chain is covalently linked by the third peptide linker sequence to the N-terminus of the VL chain.
In a more particular embodiment, the C-terminus of the sc-TCR Vxcex2 chain is covalently linked to the third polypeptide linker sequence which sequence is further linked to the N-terminus of the VH chain. Alternatively, the C-terminus of the sc-TCR V-xcex2 is covalently linked to the third polypeptide linker sequence as discussed except that the polypeptide sequence is further linked to the N-terminus of the VL chain. In another embodiment, the C-terminus of the sc-TCR V-xcex2 chain is covalently linked to a C-xcex2 chain which chain is covalently linked to the third polypeptide linker sequence which sequence is linked to the N-terminus of the VH chain. Alternatively, the C-terminus of the sc-TCR V-xcex2 chain is covalently linked to a C-xcex2 chain which chain is covalently linked to the third polypeptide linker sequence which sequence is linked to the N-terminus of the VL chain.
In a preferred embodiment, the present invention provides single-chain xe2x80x9cbispecificxe2x80x9d binding proteins that include at least one sc-TCR (or fragment thereof), and at least one sc-Fv (or fragment thereof) covalently linked together through a suitable peptide linker sequence. In instances in which more than one sc-TCR and/or sc-Fv are used the sc-TCRs and sc-Fvs are preferably the same. The single-chain bispecific binding protein will sometimes be referred to herein as a xe2x80x9cbispecific hybrid moleculexe2x80x9d or xe2x80x9csc-TCR/scFv hybrid moleculexe2x80x9d or similar phrase. The bispecific hybrid molecules of this invention may include additional amino acid sequences such as protein tags. More preferred bispecific binding proteins are discussed as follows.
For example, in one embodiment, the bispecific binding molecules include covalently linked in sequence: 1) a sc-TCR or functional fragment thereof of interest, 2) a suitable peptide linker sequence, and 3) a sc-Fv or functional fragment thereof. In a more particular embodiment, the sc-TCR further includes covalently linked in sequence: 4) the V-xcex1 chain, 5) a suitable peptide linker sequence, 6) a V-xcex2 chain, and 7) an optional C-xcex2 chain fragment. Alternatively, the sc-TCR can include covalently linked in sequence: 4) the V-xcex2 chain, 5) the linker sequence, 6) the V-xcex1 chain, and 7) an optional C-xcex2 chain or fragment thereof. In another particular embodiment, the sc-TCR further includes a C-xcex1 chain or fragment thereof covalently linked between the V-xcex1 chain and the peptide linker sequence fused to V-xcex2 chain.
In another particular embodiment, the bispecific binding molecules include a sc-Fv that which includes covalently linked in sequence: 8) the VH chain, 9) a suitable polypeptide linker sequence, and 10) the VL chain. In another embodiment, the sc-Fv includes covalently linked in sequence: 8) the VL chain, 9) the polypeptide linker sequence, and 10) the VH chain.
The single-chain polyspecific binding proteins of this invention may further include at least one protein tag covalently linked thereto, preferably from between about 1 to 3 of such tags. Preferably, the protein tag is fused to the C-terminus of a desired binding molecule although for some applications fusion to the N-terminus may be more preferred.
In a more specific embodiment, the single-chain polyspecific binding protein includes covalently linked in sequence: 1) the TCR V-xcex1 chain, 2) a peptide linker sequence, 3) the TCR V-xcex2 chain covalently linked to a C-xcex2 chain fragment, 4) a peptide linker sequence, 5) the VL chain, 5) a peptide linker sequence, and 6) the VH chain. In another embodiment, the single-chain polyspecific binding protein includes covalently linked in sequence: 1) the TCR V-xcex1 chain, 2) a peptide linker sequence, 3) the TCR V-xcex2 chain covalently linked to a C-xcex2 chain fragment, 4) a peptide linker sequence, 5) the VH chain, 5) a polypeptide linker sequence, and the 6) VL chain.
Significantly, the present invention is flexible. That is, the invention features polyspecific binding molecules that can include a variety of sc-TCR and sv-FV components. As will be appreciated, the order in which the components are made or assembled is usually not important so long as desired binding and activation characteristics are achieved.
In another embodiment, the present invention features multi-chain polyspecific binding proteins that include at least one sc-TCR (functional fragment thereof) and at least one antibody binding domain which can be, e.g., an F(v) or sc-Fv. The binding molecules more specifically include at least one xe2x80x9cjoining moleculexe2x80x9d to link the sc-TCR and antibody binding domain. As will be more fully discussed below, the joining molecule may be covalently or non-covalently linked to the sc-TCR, the antibody binding domain, or both. For example, in one preferred embodiment, two compatible joining molecules are each independently fused to the sc-TCR and the sc-Fv.
The term xe2x80x9cjoining moleculexe2x80x9d means an amino acid sequence that is capable of specifically binding, either covalently or non-covalently, to a second amino acid sequence. Sometimes the second amino acid sequence is referred to as a xe2x80x9ccognatexe2x80x9d sequence to denote capacity to form a specific binding pair. The second sequence may also be sometimes referred to herein as a second joining molecule, which second joining molecule can be the same as, or different from, the (first) joining molecule. More particular joining molecules include immunoglobulin chains and particularly constant chains (H or L) or suitable fragments thereof, coiled-coil motifs and helix-turn-helix motifs. More specific examples of joining molecules are disclosed below.
It will be apparent from the discussion which follows that in some instances a joining molecule may also serve as a protein tag.
A more particular multi-chain polyspecific binding molecule includes more than one joining molecule and preferably about 2 of such joining molecules. In a more specific embodiment, one sc-TCR is fused to the first joining molecule. The first joining molecule can be either covalently or non-covalently linked to the second joining molecule which is further linked to the antibody binding domain. However in some embodiments such as when the first and second joining molecules are suitable immunoglobulin chains, a combination of covalent and non-covalent bonds may be employed to link the sc-TCR and the antibody binding domain through the first and second joining molecules.
As an illustration, a particular multi-chain polyspecific binding protein includes covalently linked to at least one sc-TCR, preferably one sc-TCR, an immunoglobulin heavy chain (Ig-CH) or functional fragment. Sometimes this construct will be referred to herein as a xe2x80x9csc-TCR/Ig fusion proteinxe2x80x9d, xe2x80x9csc-TCR/Igxe2x80x9d or similar phrase. It will be appreciated that the immunoglobulin heavy chain portion of the sc-TCR/Ig fusion protein is representative of one type of joining molecule as defined above and in the discussion following. In a more specific embodiment, the binding molecule further includes a second joining molecule, which is preferably a suitable immunoglobulin heavy chain capable of forming a binding complex. The isotype of the immunoglobulin chains may be different but are preferably the same to facilitate binding. The second joining molecule is bound to the antibody binding domain which is preferably an F(v) and particularly an sc-Fv. In other embodiments, the sc-TCR may be further bound covalently or non-covalently to an immunoglobulin variable chain and preferably a variable light chain.
The single- and multi-chain polyspecific binding proteins disclosed herein preferably include TCR V-xcex1 and the V-xcex2 chains that are at least 90% identical to T-cell receptor V chains present on a cytotoxic T cell. Preferably, at the least the sc-TCR portion of the protein has been humanized and more preferably the entire binding protein has been humanized to enhance patient compatibility. In such embodiments it may be desirable to include at least one protein tag which can be, e.g., a detectably-labeled molecule suitable for diagnostic or imaging studies.
As will be described below, the present polyspecific binding molecules can be unmodified, or if desired, can be covalently linked to a desired molecule, e.g., drugs, toxins, enzymes or radioactive substances through a linked peptide linker sequence.
The polyspecific binding molecules of the present invention provide several significant advantages.
For example, preferred, polyspecific binding proteins are capable of associating an MHC-expressing target cell and an immune cell. That is, the present binding proteins preferably form a bridge that joins the immune cell to the MHC- or HLA-expressing cell. As noted, that association is believed to enhance recognition and facilitate damage to or killing of the target cell. In contrast, most prior immune system molecules and particularly bsFv molecules are not optimized to bind pMHC or pHLA complexes. Accordingly, the present molecules provide an effective means for killing target cells that express a pMHC or pHLA molecule.
Additionally, use of the present polyspecific binding proteins is believed to be associated with fewer adverse activities when compared to many prior immune molecules. As an illustration, many prior bsFv molecules have been reported to bind to shed TAAs. In contrast, preferred polyspecific binding molecules of this invention specifically bind TAAs in the context of MHC or HLA molecules, thereby substantially reducing or totally eliminating non-specific binding to the shed debris. Significantly, there has been much less concern in the field regarding any MHC and HLA shedding.
Further, the polyspecific binding molecules disclosed herein can bind a significantly wider spectrum of molecules than most prior recombinant immune molecules. In particular, there has been understanding that targetable antigens are often hidden inside cells making recognition and binding difficult. It is an object of the present invention to provide binding molecules that specifically bind these hidden antigens. For example, the polyspecific binding molecules include at least one sc-TCR (or functional fragment) that can bind antigens in the context of an MHC or HLA. Thus, the present binding molecules are capable of binding a large variety of antigens that are usually hidden inside cells. In contrast, most prior recombinant immune system molecules are not able to bind MHC- or HLA-presented antigens effectively.
The present invention provides still further advantages. For example, prior practice generally required extensive manipulation of TCR-related proteins (e.g., TCR receptors, TCR heterodimers, sc-TCRs), before significant amounts of protein could be obtained. In contrast, the polyspecific binding molecules of the present invention are fully soluble and can be isolated in significant quantities. Additionally, a wide variety of the polyspecific binding molecules can be presented for interaction with various immune system components such as superantigens or APCs.
Additionally, the single- and multi-chain polyspecific binding molecules include immunoglobulin chains that are readily isolated by standard immunological methods. Presence of these chains can usually facilitate detection, analysis and isolation of the binding molecules as discussed below.
In another aspect, the invention pertains to polynucleotides (RNA, mRNA, cDNA, genomic DNA, or chimeras thereof that include or consist of a sequence that encodes a single- or multi-chain polyspecific binding protein. In one embodiment, the polynucleotide includes sequence that encodes essentially all of the binding protein, e.g., as when the binding protein is a single-chain construct.
In another embodiment, the polynucleotides include a sequence that encodes a portion of the polyspecific binding protein and particularly part of certain multi-chain binding proteins discussed below. For example, a particular polynucleotide of this invention may encode an sc-TCR fused to an immunoglobulin heavy chain or suitable fragment thereof (e.g., an sc-TCR/Ig molecule). In this embodiment, the remaining part of the polyspecific binding protein can be provided in several ways. For example, it can be provided by a cell or extract thereof capable of synthesizing antibody molecules such as an antibody binding domain. The antibody or antibody-binding domain may be encoded by the cell genome or it may be encoded by an introduced DNA segment. Preferably, the cell will be an antibody-producing cell such as a hybridoma cell. Alternatively, the remaining part of the binding protein is provided by a second polynucleotide sequence that includes the DNA segment. In this embodiment, the binding protein is preferably constructed by contacting the encoded protein portions together under conditions conducive to forming the desired binding protein. As will be discussed below, the polyspecific binding proteins of this invention can be joined by one or a combination of strategies including cellular, genetic and chemical methods.
Particularly contemplated are DNA vectors that include or consist of the polynucleotides of this invention. Illustrative DNA vectors include those compatible with conventional prokaryotic or eukaryotic protein expression system. More specific examples of polynucleotides and DNA vectors.
The polynucleotides of the present advantage provide important advantages. For example, as will become apparent from the disclosure which follows, preferred polynucleotides of this invention include DNA segments that encode covalently linked scTCR and sc-Ab molecules. The DNA segments are preferably configured in a xe2x80x9ccassettexe2x80x9d format so that a segment encoding a sc-TCR or sc-Ab can be switched, as desired, with another segment encoding another scTCR or sc-Ab.
In another aspect, the present invention provides compositions and methods for selecting polyspecific binding proteins. More particularly, the compositions and methods can be employed to select sc-TCR and sc-Ab molecules with desired characteristics, thereby facilitating manufacture and use of polyspecific binding proteins that include these molecules.
In one embodiment, the invention provides recombinant bacteriophages that include at least one sc-TCR (or functional fragment) and at least one sc-Fv (or functional fragment) as fusion proteins. As will be discussed, the bacteriophages can be employed, e.g., to select sc-TCR and sc-Fv molecules for desired binding characteristics. Preferred are bispecific bacteriophages. The recombinant bacteriophages may sometimes be referred to herein as xe2x80x9cpolyfunctionalxe2x80x9d or xe2x80x9cpolyspecificxe2x80x9d to denote binding by the sc-TCR and the sc-Fv fusion proteins. The recombinant bacteriophages can be derived from well-known filamentous xe2x80x9cfdxe2x80x9d phages although related phages may be used in some cases.
More particularly, the recombinant bacteriophages of this invention include a plurality of fusion proteins that each include: 1) at least one sc-TCR or functional fragment thereof fused to a first bacteriophage coat protein, or 2) at least one sc-Ab or functional fragment thereof fused to a second bacteriophage coat protein the same or different from the first bacteriophage coat protein. Preferred bacteriophage coat proteins are essentially full-length or may be fragments thereof provided that the fragment is sufficient to display the fused molecule. By xe2x80x9cdisplayxe2x80x9d is meant that the protein fusion is part of the bacteriophage coat and is readily detectable on the bacteriophage by standard screening techniques such as those disclosed below.
In a related aspect, the invention provides a recombinant bacteriophage library that includes a plurality of recombinant bacteriophages in which each bacteriophage comprises a plurality of single-chain polyspecific binding proteins each covalently linked to a bacteriophage coat protein as a protein fusion, wherein each single-chain binding protein comprises: 1) one sc-TCR or functional fragment thereof fused to a first bacteriophage coat protein or fragment, or 2) one sc-Ab or functional fragment thereof fused to a second bacteriophage coat protein or fragment. More preferred recombinant bacteriophage libraries include bacteriophages that display bispecific binding proteins.
The recombinant bacteriophage libraries can be formatted to include a variety of TCR V chains and/or immunoglobulin variable chains. Accordingly, libraries can be used to select recombinant bacteriophages that display desired sc-TCR and sc-Ab molecules.
The recombinant bacteriophages of this invention can be isolated by a variety of conventional techniques. In one embodiment, there is provided a method for isolating the recombinant bacteriophages in which the methods include at least one and preferably all of the following steps:
a) introducing into host cells a first polynucleotide comprising a sequence encoding a first fusion protein comprising an sc-TCR covalently linked to a first bacteriophage coat protein or fragment,
b) introducing into the host cells a second polynucleotide comprising
a sequence encoding a second fusion protein comprising a sc-Fv covalently linked to a second bacteriophage coat protein or fragment.
c) culturing the host cells in medium under conditions permitting propagation of bacteriophages and display of the fusion proteins; and
d) isolating the recombinant bacteriophages from the host cell or the medium.
In a particular embodiment, the method further includes contacting an extract of the host cell or the cultured medium with a synthetic matrix capable of specifically binding one of the fusion proteins, and purifying the recombinant bacteriophage from the synthetic matrix to isolate the bacteriophage. In a more particular embodiment, the synthetic matrix includes an antibody fragment that is capable of specifically binding the recombinant bacteriophage. More specific bacteriophage isolation techniques are discussed below.
Additionally provided by the invention is a kit comprising the present recombinant bacteriophages which kit may also include suitable prokaryotic cells for propagating the bacteriophage and directions for using the kit. Also provided is a kit that includes the bacteriophage library discussed above.
The recombinant bacteriophages of this invention have additional uses and advantages. For example, the bacteriophages can be used in accord with standard screening techniques to facilitate analysis of a desired polyspecific binding molecule in vitro. More particularly, the recombinant bacteriophages can be used to assess whether a specific sc-TCR or sc-Ab such as a sc-Fv has capacity to recognize, bind and/or kill target cells of interest. Additional advantages include a relatively fast and straightforward procedure for making and testing bispecific sc-TCR/sc-Ab molecules; a short and simple purification process; and an accelerated method for testing large numbers of different hybrid molecules for efficacy in damaging or killing target cells (e.g., tumor killing).
The single- and multi-chain polyspecific binding proteins of this invention can be made as fully functional and soluble proteins by one or a combination of methods. In general, the methods involve cellular, recombinant DNA and chemical techniques, or combinations thereof.
For example, in one embodiment, there is provided a method for making a single-chain polyspecific binding protein comprising at least one sc-TCR or functional fragment thereof and at least one sc-Ab and particularly a sc-Fv or functional fragment thereof. The method includes at least one and preferably all of the following steps:
a) introducing into a host cell a DNA vector encoding a single-chain polyspecific binding protein of interest,
b) culturing the host cell in media under conditions sufficient to express the single-chain polyspecific binding protein in the cell or the media; and
c) isolating the single-chain polyspecific binding protein from the cell or media.
Additionally provided are methods for making a multi-chain polyspecific binding protein comprising at least one sc-TCR or functional fragment thereof and an antibody binding domain or functional fragment. In one embodiment of the method, the antibody-binding domain is a F(v). In particular, a cell or cell extract is used to form at least part of the multi-chain binding protein. More particularly, an antibody producing cell such as a hybridoma is employed. In one embodiment, the method includes at least one and preferably all of the following steps:
a) introducing into a hybridoma cell a DNA vector encoding at least one sc-TCR, preferably one sc-TCR (or functional fragment) covalently linked to an immunoglobulin constant heavy chain or fragment thereof,
b) culturing the hybridoma cell in media under conditions conducive to forming a specific binding complex between the immunoglobulin constant heavy chain or fragment encoded by the DNA vector and immunoglobulin chains produced by the hybridoma; and
c) purifying the multi-chain polyspecific binding protein from the hybridoma cells or media.
In a more specific embodiment, the method provides for a multi-chain polyspecific binding protein that includes an immunoglobulin variable light chain covalently linked to the sc-TCR, i.e., a bispecific binding protein.
The present invention provides additional methods for making the multi-chain polyspecific binding proteins. For example, in one embodiment, each chain of a desired binding protein is made independently, e.g., by recombinant DNA or chemical methods. Preferably, the binding protein further includes at least one joining molecule, preferably two joining molecules the same or different. In a particular embodiment, the method includes at least one and preferably all of the following steps:
a) providing a first sequence that includes at least one sc-TCR or functional fragment thereof covalently linked to a first joining molecule,
b) contacting the first sequence with a second sequence that includes at least one sc-Fv or functional fragment thereof linked to a second joining molecule, wherein the contacting is under conditions sufficient to form a specific binding complex between the first and second joining molecules; and
c) forming the multi-chain polyspecific binding protein. Preferably, the multi-chain polyspecific binding protein is bispecific.
More specific recombinant DNA and chemical methods for making the multi-chain polyspecific binding proteins are disclosed below.
As discussed, the present polyspecific binding proteins also have significant uses in vivo. For example, the binding proteins can be used to redirect the specificity of certain immune cells, e.g., to eliminate target cells such as virally-infected or tumor cells. In some instances, the tumor cells may also be virally-infected. As discussed, preferred use of the present binding molecules can increase damage or elimination of the target cells. Accordingly, the present invention can be used in vivo to kill target cells by enhancing immune system activation against those target cells. Preferred in vivo use of the present polyspecific binding molecules includes use in a mammal such as a rodent, primate or domesticated animal, and especially a human patient.
Thus, in one aspect, the invention provides methods for damaging or preferably killing a target cell comprising an MHC or HLA of interest. In one embodiment, the method includes at least one and preferably all of the following steps:
a) contacting a plurality of cells with a polyspecific binding protein, wherein the plurality of cells comprises immune cells comprising an antigen and target cells comprising the MHC or HLA,
b) forming a specific binding complex (bridge) between the MHC or HLA on the target cells and the antigen on the immune cells sufficient to activate the immune cells; and
c) killing the target cells with the activated immune cells. It will be appreciated that by the term xe2x80x9cactivatedxe2x80x9d is meant that the immune cells are capable of damaging or killing the target cell as determined, e.g., by cytokine and cytotoxicity assays described below.
If desired, the above-described method may be conducted in vitro such as in a cell culture dish.
The single- and multi-chain polyspecific binding proteins of this invention have additional uses in vitro and in vivo.
For example, preferred polyspecific binding molecules of this invention can be used in vitro or in vivo to detect and preferably form a bridge between target cells and immune cells. Formation of the bridge can be used to isolate cells expressing desired MHC, HLA or antigen markers.
The present polyspecific binding proteins also find use in the detection and analysis of MHC or HLA molecules and cell surface antigens. The present binding proteins can also be used for diagnostic purposes such as for the detection of immune system cells and especially T-cells with pathogenic properties. The present binding molecules can additionally be employed in functional, cellular and molecular assays, and in structural analysis, including X-ray crystallography, nuclear magnetic resonance imaging, computational techniques such as computer graphic display.
In another aspect, the present invention further provides treatment methods for reducing or eliminating presence of the target cells in a mammal. In particular, the methods include administering a polyspecific binding protein of this invention in a pharmaceutically acceptable formulation. If desired, the sc-TCR or sc-Ab portion of particular polyspecific binding molecule can be removed prior to or during the administration to facilitate a specific treatment method.
In a more particular embodiment, the treatment methods are employed to treat cancer and a viral infection. In particular, the methods include administering a therapeutically effective amount of at least one polyspecific binding protein of this invention to a mammal and especially a human patient. Preferably the amount is sufficient to treat the cancer and/or the viral infection. The methods may be used to treat an existing condition or may be used prophylactically as needed. The present treatment methods may be used alone or in combination with other therapies if desired.
Preferred treatment methods of the invention reduce or eliminate presence of specific target cells in a mammal, particularly a rodent or a primate such as a human. In one embodiment, the treatment methods include obtaining an sc-TCR or sc-Ab from the polyspecific binding molecule (e.g., by protease treatment). The sc-TCR or sc-Ab so obtained may be administered to the mammal instead of or in conjunction with the polyspecific binding molecule. For most applications involving animal use, it will be preferred to minimize undesired immune responses against the present binding molecules, e.g., by using immunoglobulin chains of a haplotype compatible with the animal being used.